Differential roles of PKC-theta in the regulation of intracellular calcium concentration in primary T cells

J Mol Biol. 2006 Jan 20;355(3):347-59. doi: 10.1016/j.jmb.2005.10.043. Epub 2005 Nov 8.

Abstract

Activation of T lymphocytes requires protein kinase C theta (PKC-theta) and an appropriately elevated free intracellular Ca2+ concentration ([Ca2+]i). Here, we show that phorbol 12 myristate 13-acetate (PMA) inhibited Ca2+ influx in wild-type but not PKC-theta-/- T cells, suggesting that PKC-theta plays a role in PMA-mediated inhibition of Ca2+ influx. In contrast, T cell receptor (TCR) crosslinking in the same PKC-theta-/- T cells did result in significantly decreased [Ca2+]i compared to wild-type T cells, suggesting a positive role for PKC-theta in TCR-mediated Ca2+ mobilization. In PKC-theta-/- mice, peripheral mature T cells, but not developing thymocytes, displayed significantly decreased TCR-induced Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation upon sub-optimal TCR crosslinking. The decreased intracellular free Ca2+ was due to changes in Ca2+ influx but not efflux, as observed in extracellular and intracellular Ca2+ mobilization studies. However, these differences in Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation disappeared with increasing intensity of TCR crosslinking. The enhancing effect of PKC-theta on Ca2+ influx is not only dependent on the strength of TCR crosslinking but also on the developmental stage of T cells. The underlying mechanism involved phospholipase Cgamma1 activation and inositol triphosphate production. Furthermore, knockdown of endogenous PKC-theta expression in Jurkat cells resulted in significant inhibition of TCR-induced activation of NFAT, as evidenced from NFAT reporter studies. Forced expression of a constitutively active form of calcineurin in PKC-theta-/- Jurkat cells could readily overcome the above inhibition. Thus, PKC-theta can both positively and negatively regulate the Ca2+ influx that is critical for NFAT activity.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcineurin / metabolism
  • Calcium / metabolism*
  • Cell Differentiation
  • Cross-Linking Reagents / metabolism
  • Enzyme Activation
  • Humans
  • In Vitro Techniques
  • Inositol Phosphates / metabolism
  • Isoenzymes / genetics
  • Isoenzymes / physiology*
  • Jurkat Cells
  • Mice
  • Mice, Knockout
  • NF-kappa B / metabolism
  • NFATC Transcription Factors / metabolism*
  • NFATC Transcription Factors / physiology
  • Phospholipase C gamma / metabolism
  • Protein Kinase C / genetics
  • Protein Kinase C / physiology*
  • Protein Kinase C-theta
  • Protein Transport
  • Receptors, Antigen, T-Cell / metabolism
  • T-Lymphocytes / cytology
  • T-Lymphocytes / drug effects
  • T-Lymphocytes / metabolism*
  • Tetradecanoylphorbol Acetate / pharmacology
  • Transcription Factor AP-1 / metabolism

Substances

  • Cross-Linking Reagents
  • Inositol Phosphates
  • Isoenzymes
  • NF-kappa B
  • NFATC Transcription Factors
  • Receptors, Antigen, T-Cell
  • Transcription Factor AP-1
  • inositol 1,3-bisphosphate
  • PRKCQ protein, human
  • Prkcq protein, mouse
  • Protein Kinase C
  • Protein Kinase C-theta
  • Calcineurin
  • Phospholipase C gamma
  • Tetradecanoylphorbol Acetate
  • Calcium